1. Field of the Invention
The present invention relates to an in-vitro translation system capable of extracellularly synthesizing protein by employing a cell extract, and particularly to a system capable of implementing both protein synthesis and glycosylation, to synthesize glycoprotein from the cell extract.
2. Description of the Related Art
Functional information within living organisms is recorded on nucleic acids. Proteins (which are functional molecules) are translated, and functional RNA molecules (ribozymes for example) are transcribed, using this nucleic acid as the template. In recent years, analysis of nucleic acid and protein supporting biofunction has been actively conducted, and development of analyzing methods and analyzing means has also been promoted.
Methodology for analyzing nucleic acid has particularly shown impressive progress pursuant to the development of a polymerase chain reaction (PCR) and the like. According to PCR, by adding a primer and template DNA in a cell-free reaction solution containing polymerase enzymes, it is possible to freely amplify DNA fragments of template DNA. In other words, the nucleic acid can be freely synthesized and amplified extracellularly. The synthesized nucleic acid can be, for example, used to determine the primary structure (base sequence), and it is thereby possible to accelerate the progress of nucleic acid analysis, such as genome analysis.
Meanwhile, with respect to methodology for analyzing protein, various cell-free translation systems have been developed since A. S. Spirin, et al., developed an in-vitro protein synthesis system employing E. coli extract. In addition to the aforementioned E. coli, there are cell-free translation systems which utilize other material, for example, cell extracts prepared from wheat germ, rabbit reticulocyte, and so on.
Among the above, the more general cell-free translation system deriving from wheat germ grinds the wheat germ together with glass beads, a mortar, or the like and synthesizes protein from mRNA by employing the cell extract obtained from such ground wheat germ. In other words, it is possible to recover the cell extract from wheat germ while preserving the protein synthesis (translation) activity existing in the wheat germ, and protein may be freely synthesized extracellularly by employing such recovered cell extract.
If protein can be freely synthesized extracellularly as described above, it becomes possible to obtain a desired protein easily by eliminating complex factors and complications that accompany synthesizing the protein in cells, and this is advantageous in terms of analyzing the protein. From the foregoing perspective, improvement of cell-free translation systems has been conducted heretofore, and such technology is disclosed, for example, in Japanese Patent Publication No. H1-503119, Japanese Patent Laid-Open Publication No. H4-200390, Japanese Patent Laid-Open Publication No. H7-203984, among others. Moreover, such cell-free translation systems are commercially available as kits (Amasham, etc.) and are widely available.
Nevertheless, although the conventional cell-free translation systems described above are capable of performing translation to the protein, there is a problem in that they are not able to perform post-translational modification of the translated protein. In other words, it is known that many of the intracellular proteins are translated as protein based on the mRNA transcribed from the template nucleic acid, and modified after such translation. Glycosylation is one such post-translational modification.
The sugar chain added pursuant to post-translational glycosylation is considered to function as a signal or ligand relating to the recognition or adhesion between substances or cells, as a function-adjusting factor of the protein itself, or as a protective or stability factor of the protein. Thus, in order to analyze the function within living organisms with respect to the protein being glycosylated, it is necessary to obtain such a glycosylated protein.
This glycosylation adds a sugar chain to a specific amino acid of the protein. Since the glycosylation reactions differ variously and are complicated, it is not easy to chemically add a sugar chain to a protein synthesized with the foregoing cell-free translation systems.
In view of such a problem, a currently available biochemical method, for example, derives an extract having glycosylation activity from dog tissue and uses the extract to add a sugar chain to protein from a cell-free translation system. The extract having glycosylation activity is prepared by crushing the dog tissues with a homogenizer and recovering microsome fractions containing a Golgi body by centrifugation.
This dog tissue extract, however, is used separately from conventional cell-free translation systems. Specifically, protein is synthesized with a cell-free translation system, and, after having recovered the synthesized protein, glycosylation is performed thereto by transferring the synthesized protein to such dog tissue extract. As a result of this acquirement of dog tissue extract, the extracellular biochemical synthesis of glycoprotein became possible. And, by employing this synthesized glycoprotein for the likes of protein performance analysis, analysis capable of further reflecting the intercellular reaction, as opposed to protein synthesized with a conventional cell-free translation system in which glycosylation has not been performed thereto, is anticipated.
Nevertheless, with the glycoprotein synthesis employing the conventional dog tissue extract, glycosylation is conducted after recovering the protein that has been synthesized with a conventional cell-free translation system. As described above, synthesis of glycoprotein through separate use of a cell-free translation system and a glycosylation system is not preferable in proteins that generally denature easily, and it is also possible that this will lead to deterioration in activity. Further, in addition to the physical influence on the protein, people working with such proteins have to concentrate on preparing the aforementioned two systems and synthesizing glycoprotein in two stages, thus making the procedure complicated.
Moreover, with respect to a cell extract capable of performing glycosylation, only those deriving from a restricted tissue such as dog tissue can be used at present, and it is not yet possible to recover a glycosylation activity from universal tissue cells. The type of sugar chain will differ depending on the type of cell, and it is anticipated that the glycosylation reaction will differ depending on such cell type. Therefore, protein glycosylation can be freely designed if the recovery of glycosylation activity from various cells becomes possible.
Furthermore, in recent years, various protein preparations have been developed in the medical field, and it is known that the effect of such preparations is influenced by the existence or type of sugar chain of the constituent protein. Thus, the realization of recovery of glycosylation activity from various cells is anticipated to also contribute significantly to the development and improvement of such protein preparations.
Therefore, in view of the foregoing problems, the inventors of the present application conducted intense study regarding the preparation of a cell extract capable of conducting a series of processes from protein synthesis to glycosylation within a single system, and, through this research, they realized a novel preparation of a cell-free extract differing from a conventional cell-free translation system, and enabled the series of processes from protein synthesis to glycosylation to be conducted within a single system by employing such extract.